HIV-1 infection is a major global health problem for which the favored solution is an effective vaccine. To date, experimental HIV-1 vaccines have typically elicited poor neutralizing antibody responses, which has been attributed in part to non-native presentations of the envelope glycoproteins (Env). Nevertheless, broadly neutralizing antibodies against primary isolates of HIV-1 do exist, and there is strong evidence that such neutralizing antibodies could play an important role in preventing HIV-1 infection. The objectives of this proposal are two-fold. One objective is to determine the X-ray crystal structure of a full-length gp120 molecule that will provide much needed structural information and can in Env-based vaccine design. The second objective is to identify, and develop different presentations of, novel HIV-1 Env variants that can elicit broadly neutralizing antibody responses against primary isolates of HIV-1. Both objectives take advantage of diverse, recombined Env libraries generated in vitro. Specifically, we will use modern high-throughput methods to obtain high-resolution structural information on a recombined full-length gp120 variant alone, and/or in complex with the broadly neutralizing antibody against HIV-1, b12. A number of different display formats of the Env molecule will be evaluated for improved immunogenicity in animals relative to unmodified and wild-type JR-CSF Env. In particular, screens for thermostable, native Env trimers will be performed and the immunogens tested. In addition, use of a covalently-stabilized trimerization scaffold, the virus-like particle format and immunofocusing techniques will be evaluated for immunogenicity in several different Env backgrounds. Finally, we will determine if sera, identified as having HIV-1 neutralizing activity in a pseudotype assay format, also neutralize primary HIV-1 virions in a conventional PBMC assay in order to identify the optimal immunogens. The iterative and rationally-guided screening process provides a novel and potentially powerful approach to HIV-1 vaccine design.